Mechanism and Inhibition of the Plasmid-Mediated Colistin Resistance Determinant MCR-1

该项目旨在了解细菌抵抗粘杆菌素作用的一种机制，粘杆菌素是一种肽抗生素，可破坏革兰氏阴性菌（如大肠杆菌）的外膜。大肠杆菌，当遇到对其他药物的耐药性时，将其用作最后的治疗手段。对粘菌素的耐药性以前是已知的，但很少见;然而，我们的研究小组最近在大肠杆菌中发现了质粒介导的粘菌素耐药性。大肠杆菌菌株来源于中国的养殖猪。负责的基因mcr-1已经在来自兽医，环境和人类样本以及世界各地的多种细菌中被鉴定出来。MCR-1通过修饰细菌的外膜来保护细菌免受粘菌素的侵害;具体而言，它编码一种膜结合酶，该酶催化磷酸乙醇胺转移至脂多糖的脂质A组分。该反应的机理仍有待探索。最近，我们已经成功地获得了可溶性的晶体结构，MCR-1酶的催化域，揭示这是一个锌金属酶，并提出了一个催化机制，涉及一个单一的锌离子。这不同于先前关于涉及两个或三个金属离子的相关酶的机制建议。因此，我们现在试图探索MCR-1的机制，使用计算和实验方法的组合，并随后利用我们的发现来识别酶的抑制剂。基于我们的结构，学生将使用专业的生物分子模拟方法（分子动力学和量子力学/分子力学（QM/MM））来研究MCR-1锌中心及其与磷酸乙醇胺的相互作用。结果将生成MCR-1机制的模型，将使用结构（X射线晶体学）、生物物理（光谱学）、生物化学（磷酸乙醇胺转移试验）和微生物（重组大肠杆菌中粘菌素敏感性试验）的组合进行实验检测。coli）方法。随后，这一信息将用于计算研究，旨在鉴定能够结合MCR-1活性位点并因此破坏其对细菌脂质A作用的小分子。这些将确定一组潜在的MCR-1抑制剂，这些抑制剂将在体外进行实验测试，并测试它们对粘菌素杀死产生MCR-1的细菌的影响。这是一个多学科的项目，我们预计学生将在奖励期间的大部分时间里参与实验室和计算工作。学生将获得最先进的计算方法和广泛的实验方法的培训，以表征生物大分子，它们的功能和相互作用。

This project seeks to understand one mechanism by which bacteria resist the action of colistin, a peptide antibiotic that disrupts the outer membrane of Gram-negative bacteria such as E. coli and that is used as a last resort treatment when resistance is encountered to other drugs. Resistance to colistin was previously known, but rare; however our team recently discovered plasmid-mediated colistin resistance in an E. coli strain originating from a farmed pig in China. The gene responsible, mcr-1, has since been identified in multiple bacteria from veterinary, environmental and human samples and in worldwide locations. MCR-1 protects bacteria from colistin by modifying their outer membranes; specifically it encodes a membrane-bound enzyme that catalyses transfer of phosphoethanolamine to the lipid A component of lipopolysaccharide. The mechanism of this reaction remains to be explored.Recently we have succeeded in obtaining a crystal structure for the soluble, catalytic domain of the MCR-1 enzyme, revealing this to be a zinc metalloenzyme and suggesting a catalytic mechanism involving a single zinc ion. This differs from previous mechanistic proposals for related enzymes that involve two or three metal ions. Hence we now seek to explore the mechanism of MCR-1 using a combination of computational and experimental approaches, and subsequently to exploit our findings to identify inhibitors of the enzyme. Based upon our structure the student will use expert biomolecular simulation methods (molecular dynamics and quantum mechanics/molecular mechanics (QM/MM)) to investigate the MCR-1 zinc centre and its interactions with phosphoethanolamine. The results will generate a model for MCR-1 mechanism that will be tested experimentally using a combination of structural (X-ray crystallography), biophysical (spectroscopy), biochemical (assays of phosphoethanolamine transfer) and microbiological (assays of colistin susceptibility in recombinant E. coli) approaches. Subsequently this information will be exploited in computational studies aimed at identifying small molecules able to bind the MCR-1 active site and that thus disrupt its action upon bacterial lipid A. These will identify a panel of potential MCR-1 inhibitors that will be tested experimentally in vitro and for their effects upon colistin killing of MCR-1 producing bacteria.This is a multidisciplinary project where we anticipate that the student will be involved in both laboratory and computational work for the majority of the award period. The student will gain training in state of the art computational methods and a wide range of experimental approaches to characterising biological macromolecules, their function and interactions.